This invention relates generally to a thermal cycling device and, more specifically, to an apparatus for the rapid heating and cooling of liquid samples.
A thermal cycling device is an apparatus used to continuously change the temperature of a liquid sample. As used herein, the term xe2x80x9cliquidxe2x80x9d refers to pure liquids, as well as liquids containing particulate matter (especially biological material) and solvents containing solute.
Thermal cycling devices are well-known in the art and specific embodiments have been described in the scientific and patent literature. These devices fall into two general categories.
The first category is a system based on the heating or cooling of a metal block typically either by moving a fluid through the block or by the addition of peltier heating directly to the block. A number of individual plastic tubes, a well plate (or a microliter plate which consists, essentially, of a number of plastic tubes connected together in a rectangular array) are used to hold the liquid samples. The plastic tubes are placed in the block and the temperature of the sample is regulated by changing the temperature of the metal block. The rate at which the temperature can be changed is limited by the relatively large thermal mass of the metal block. This being the case, the maximal rate of temperature change is relatively slow due to the fact that heat has to be added or removed from a relatively large thermal mass and this mass takes a significant time to reach thermal equilibrium. A second disadvantage of this category of thermal cyclers is that the liquid samples to be heated or cooled are xe2x80x9cinsulatedxe2x80x9d from the block by the plastic wall of the sample container (i.e., by the plastic tubes). Since plastic is a good insulator, not only must the heating and cooling source overcome the thermal mass of the metal block, but it must also overcome the insulating properties of the plastic sample container.
The sample containers currently in use for polymerase chain reaction (PCR) and Cycle Sequencing are either thin walled microfuge tubes, thin walled microwell platesxe2x80x94usually either 96-well or 384-wellxe2x80x94or microcapillary tubes. All of these containers suffer from a common problem in that in all cases, the samples are heated or cooled from an external source and the heating and cooling system must overcome the insulating properties of the container.
A related problem with these devices is that the plastic tubes are quite large in comparison to the liquid sample. For example, when a 96-well plate or 384-well plate is used in these systems, the container volume ranges from 300 microliters (xcexcl) to 120 xcexcl whereas the sample volume is usually only 5-25 xcexcl. When a sample is heated during a polymerase chain reaction (PCR) cycle, the liquid will evaporate and condense on the interior wall surfaces of the sample container. Loss of liquid from the sample will change the concentrations of the reaction components and lead to spurious results.
Specific measures have been taken to overcome the serious problem of evaporation and condensation. One solution is to place a lid over the sample container or well plate. A heated well-plate is placed in contact with the sample container lid and heated to 20-30xc2x0 C. higher than the sample temperature. This will minimize the condensation of the liquid on the walls and surface of the container but does nothing for the total evaporation since the air volume within the sample container is large and can hold a significant amount of liquid vapor (from 0.06-0.15 xcexcl), especially at these elevated temperatures. In a 25 xcexcl reaction volume, this liquid loss may not have a significant effect, however, when the reaction volume approaches 1 xcexcl, a loss of 15% of the liquid volume will adversely affect the reaction.
A number of manufacturers currently produce thermal cyclers that utilize a metal block for heating and cooling liquid samples. These include MJ Research, Techne, Lab Line, Thermolyne, Corbett Research, and Hybaid.
The second type of thermal cycling device involves the use of microcapillary tubes that are placed in a chamber, and heating the chamber by forcing hot air or cold air into the chamber. This method is somewhat similar to heating the samples with a convection oven and cooling the samples with a refrigeration system. A current manufacturer of this type of thermal cycler is Idaho Technologies.
This second type of thermal cycling device has the advantage that the thermal mass that needs to be heated is relatively small, i.e, the capillary tube, sample, and the interior of the chamber. However, this system has a number of limitations. First, the samples need to be sealed within a glass capillary tube. This requires that the sample be drawn into the tube via capillary action then the end of the tube has to be sealed with a flame hot enough to melt the glass. Capillary tubes by their very nature are difficult to manipulate and are not suitable to robotic automation. That is, while a limited number of samples can be prepared in this fashion, it would be extremely difficult, if not impossible to process the number of samples per day (usually on the order of 100,000 that are usually necessary for a particular study). Second, glass is a fairly good insulator so, similar to the problem described above with the plastic tubes, this system is also limited in that in order to heat or cool the sample, the insulating properties of the container must be overcome.
One of the biggest drawbacks of the commonly used polypropylene microwell plates is that the material changes shape upon repeated heating and cooling cycles. For example, upon heating a standard polypropylene microwell plate to 95xc2x0 C. and cooling back to room temperature, the plastic may deform by as much as 1 cm from corner to corner. The result of this is that the plate cannot be used directly on standard laboratory automationxe2x80x94for example, a 96 or 384 channel pipettor would hit the bottom of the wells on one corner of the plate yet remain up to 1 cm from the bottom of the other corner of the plate. To compensate for this, the samples are typically moved, singly, into a plate that has not been subjected to thermal cycling or the plate is forced into a retaining device that will hold the plate in the proper shape.
Microcapillaries as a container for PCR and Cycle Sequencing have an associated set of problems. First and foremost is the fact that these containers are not automation friendly. That is, it is difficult if not impossible for robotic liquid handlers to place liquid within a capillary. Consequently, the liquid handling is typically performed in a microfuge tube or microwell plate; then the capillary is brought into contact with the liquid and the liquid is drawn into the capillary by capillary action. This negates one of the presumed advantages of the capillary system in that a significantly larger volume of reagent must be prepared even though only a small portion of that reagent is used within the capillary. Another problem with capillaries is that they are difficult to seal. Each capillary typically has to be heat sealed to melt the glass capillary. Even if other types of materials are used for the capillaries, sealing is difficult and not amenable to automation. Finally, after the reaction has taken place, the capillary must be broken or cut and the reaction product removed. Since the sample is held within the capillary by capillary action, the removal of the sample is difficult at best.
In order to increase throughput and decrease the volume of the reaction, there is a desire to move from 384-well plates to 1536-well plates. This will allow a four-fold increase in density and throughput and, because the volume of the well is much smaller (75 microliters in a 384-well plate vs. xe2x88x926 microliters in a 1536 well plate) the reactions can be performed at 1 microliter vs. the typical 5-25 microliters that is currently performed in 384-well plates. The miniaturization from a 384-well system to a 1536-well system will yield about a 5-25 fold reagent savings. These two advancements over the current methods will yield nearly a 100-fold improvement in reagent savings and throughput.
Commercially available 1536-well plates are not conducive to the current methods of thermal cycling. There are several reasons for this. With a very high well density and a center to center spacing of 2.25 millimeters the inter-well distance is approximately 0.5 millimeters. This small distance makes it nearly impossible to surround each well with a heating/cooling unit as is currently done in 96-well or 384-well systems. Also, the plastic surface area to volume ratio is approximately 7-fold higher than in a 96-well plate or 384-well plate. With the increased plastic area, the insulating properties of the plastic that comprises the well is very difficult to overcome. Heating the plate from just the bottom is not practical since this causes temperature gradients within the well and consequently non-uniform heating.
Typically, PCR and other methods have involved placing the samples in a small microtube and then placing the microtube in a water bath or heat block for temperature regulation. This method of controlling the temperature of the reaction has been successfully used on single tubes, 96-well plates, and 384-well plates. However, as the well density increases, it becomes difficult to surround each well with uniform temperature control. Moreover, plastics typically act as excellent insulators so the external heating and cooling system has to overcome the insulating properties of the plastic before an effect on the solution is observed. An additional problem is that the solution volume is very small in comparison to the total well volume. Consequently, when the well is heated, the solution tends to evaporate and then condense on the cover of the well. This causes the concentration of the various components in the well to change and can adversely affect the reaction. To compensate for this problem, many systems heat the covers so that condensation on this surface is limited. An additional problem is that as the well density increases from 96-well plates to 384-well plates, the plates tend to warp and become misshapen during the heating and cooling cycles. This warping of the plates makes it difficult for them to be handled effectively by robotic or automated systemsxe2x80x94an absolute requirement for high throughput.
The present invention can be divided into two embodiments. The first embodiment consists of a device for heating and cooling a lid that is designed to fit over a well plate. There are two variations for this lid heating/cooling system. First, one air source passing over a heating coil and another air source passing over a cooling unit (air conditioning) are proportionally mixed in order to regulate the temperature. The mixed air is directed via a series of ducts such that it heats or cools the surface of the lid in a uniform and highly controlled fashion. The second method is to bring a heating/cooling source, such as a Peltier device or heat/cold block in direct contact with the surface of the lid. The large thermal mass of the heat/cold block will rapidly add or remove heat from the samples via the pins inserted into the sample.
The second embodiment is a device, in particular a well-plate containing, for example, 1536-wells with each well having a volume of approximately 6 xcexcl, and a mating plate lid. The lid to this plate may have a copper clad surface and may contain a rubber seal on the other surface. Protruding through this lid is a series of xe2x80x9cpinsxe2x80x9d that extend approximately from the rubber surface and which communicate with the copper clad surface for transferring heat from the lid. The device in the first embodiment heats or cools the copper clad surface and pins of the device in the second embodiment. The copper clad surface and pins rapidly transfers heat or removes heat from the pins. The pins in turn transfer heat or remove heat from the sample.
The present invention describes a device whereby the wells of a 1536-well plate, for example, can be uniformly heated by applying a heating or cooling source directly into the well via the lid; the pins are either in direct contact with the liquid sample(s) or near the liquid sample(s) such that it can conduct heat into or out of the sample.